Glutamic acid derivatives

ABSTRACT

All isomeric forms and mixtures of isomers of glutamic acid compounds of the formula ##STR1## wherein the glutamic acid of D- or L- configuration, R 1  is selected from the group consisting of hydrogen, alkyl of 1 to 5 carbon atoms, an amino acid, a peptide of 2 to 4 amino acids and an amino acid or a peptide of 2 to 4 amino acids in which the amine is esterified with an optionally unsaturated aliphatic carboxylic acid of 6 to 24 carbon atoms or R 1  is selected from the group consisting of a residue of a C 6  -C 24  optionally unsaturated aliphatic acid, R 5  is selected from the group consisting of hydrogen or an alkyl radical of 1 to 5 carbon atoms, R 3  is selected from the group consisting of hydroxy, alkoxy of 1 to 5 carbon atoms, an amino acid with the amine optionally substituted with alkyl of 1 to 5 carbon atoms, Z is ##STR2## R 2  is selected from the group consisting of hydrogen, an amino acid and a peptide of 2 to 4 amino acids, R 4  is selected from the group consisting of hydroxy, alkoxy 1 to 5 carbon atoms and an amino acid optionally substituted on the amine with alkyl of 1 to 5 carbon atoms, U is selected from the group consisting of ##STR3## --CH═CH--CH 2  -- (E or Z isomer), --CH 2  --CH═CH-- (E or Z isomer) and ##STR4## or U and Y together are ═CH--CH 2  --CH 2  -- (E or Z isomer) and X is hydrogen or U and X together are ═CH--CH 2  --CH 2  -- (E or Z isomer) and Y is hydrogen and their salts with non-toxic, pharmaceutically acceptable acid or bases having immunomodulatory properties.

PRIOR APPLICATION

This is a continuation-in-part application of copending application Ser.No. 161,163 filed Feb. 26, 1988.

STATE OF THE ART

French Patent No. 2,566,410 describes diaminopimelic acid compoundshaving antibacterial properties and U.S. Pat. No. 4,311,640 describesrelated compounds.

OBJECTS OF THE INVENTION

It is an object of the invention to provide the novel compounds offormula I and their non-toxic, pharmacutically acceptable addition saltsand a process for their preparation.

It is another object of the invention to provide novel immunomodulatingcompositions and a novel method of inducing immunomodulating activity inwarm-blooded animals.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel compounds of the invention are selected from the groupconsisting of all isomeric forms and mixtures of isomers of glutamicacid compounds of the formula ##STR5## wherein the glutamic acid is ofD- or L- configuration, R₁ is selected from the group consisting ofhydrogen, alkyl of 1 to 5 carbon atoms, an amino acid, a peptide of 2 to4 amino acids and an amino acid or a peptide of 2 to 4 amino acids inwhich the amine is esterified with an optionally unsaturated aliphaticcarboxylic acid of 6 to 24 carbon atoms or R₁ is selected from the groupconsisting of a residue of a C₆ -C₂₄ optionally unsaturated aliphaticacid. R₅ is selected from the group consisting of hydrogen and an alkylof 1 to 5 carbon atoms, R₃ is selected from the group consisting ofhydroxy, alkoxy of 1 to 5 carbon atoms and an amino acid with the amineoptionally substituted with alkyl of 1 to 5 carbon atoms, Z is ##STR6##R₂ is selected from the group consisting of hydrogen, an amino acid anda peptide of 2 to 4 amino acids, R₄ is selected from the groupconsisting of hydroxyl, alkoxy of 1 to 5 carbon atoms and an amino acidoptionally substituted on the amine with alkyl of 1 to 5 carbon atoms, Uis selected from the group consisting of ##STR7## --CH═CH--CH₂ -- (E orZ isomer), --CH₂ -CH═CH-- (E or Z isomer) and ##STR8## or U and Ytogether are ═CH--CH₂ --CH₂ -- (E or Z isomer) and X is hydrogen or Uand X together are ═CH--CH₂ --CH₂ -- (E or Z isomer) and Y is hydrogenand their salts with non-toxic, pharmaceutically acceptable acid orbases.

The amino acid in the compounds of formula I is preferably an α-aminoacid and examples of such acids are Ala, Val, Ival, Leu, Ile, Asp, Asn,Glu, Gln, Ser, Thr, Cyc, Met, Lys, Arg, Phe, Tyr, Trp, His and Pro, Nva,Nle, Hyp, Orn, In the D or L form, as well as Sar and Gly, it beingpossible for all the said acids to be N-esterified or N-alkylated. Inthe case of a peptide of 2, 3 or 4 amino acids, the latter are chosenfrom the group consisting of the above amino acids. It will beunderstood by convention that the symbols for the α-amino carboxylicacids denote these acids in their D or L configuration (for example, theterm Ala denotes alanine in D form or in L form).

The saturated or unsaturated aliphatic acid of 6 to 24 carbon atoms,preferably from 12 to 22 carbon atoms, may be stearic acid, palmiticacid, lauric acid, caprylic acid, myristic acid, α- or γ-linolenic acid,linoleic acid, arachidonic acid or docosapentaenoic acid.

Examples of alkyl of 1 to 5 carbon atoms are pentyl, isobutyl, butyl andisopropyl, and preferably propyl, ethyl or methyl. Examples of alkoxy of1 to 5 carbon atoms are pentoxy or butoxy, but preferably propoxy,ethoxy or methoxy.

The products of formula I contain one or more asymmetric carbon atomsand, as stated above, the subject of the invention is the said compoundsof formula I in all their possible isomeric forms and in the form ofmixtures.

The non-toxic, pharmaceutically acceptable salts of the derivatives ofthe invention can be formed with organic and inorganic bases such asalkali metal and alkaline earth metal hydroxides such as, for example,sodium, potassium, lithium and calcium hydroxides and magnesium orammonium hydroxide. Among organic bases, there may be mentionedsubstituted or unsubstituted alkylamines such as trimethylamine,methylamine, propylamine, N,N-dimethylethanolamine ortris(hydroxymethyl)methylamine. Basic amino acids such as lysine orarginine may also be mentioned as well as glucosamine or procaine may bementioned.

The addition salts with inorganic or organic acids can be, for example,the salts formed with hydrochloric acid, hydrobromic acid, nitric acid,sulfuric acid, phosphoric acid, acetic acid, formic acid, propionicacid, maleic acid, fumaric acid, succinic acid, tartaric acid, citricacid, oxalic acid, glyoxylic acid and aspartic acid, alkanesulfonicacids such as methane- and ethanesulfonic acid, arylsulfonic acids suchas benzene- or p-toluenesulfonic acid, and arylcarboxylic acids such asbenzoic acid.

Among the preferred compounds of formula I are those wherein theglutamic acid has the D-configuration, those wherein R₃ and R₄ arehydroxy, those wherein R₅ is hydrogen and their non-toxic,pharmaceutically acceptable acid addition salts. Specific preferredcompounds are 2-amino-6-γ-D-glutamylamino)-4-methyleneheptanedioic acid,2-(L-alanylamino)-6-(γ-Dglutamylamino)-4-methyleneheptanedioic acid,2-amino-4-methylene-6-[{N-(N-1-oxo-octadecyl)-L-alanyl-γ-D-glutamyl}-amino]heptanedioicacid, 2-amino-4-methylene-6 [[N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioic acid and their non-toxic,pharmaceutically acceptable acid addition salts.

The novel process of the invention for the preparation of the compoundsof formula I comprises reacting a glutamic acid compound of the formula##STR9## wherein Alk is alkyl of 1 to 3 carbon atoms and R₁ ' has thedefinition of R₁ other than hydrogen or an amino protective group with acompound of the formula ##STR10## as an individual isomer or mixture ofisomers wherein U has the above definition, Alk₁ and Alk₂ are alkyl of 1to 3 carbon atoms, X' and Y' are hydrogen or --COOAlk₃, Alk₃ is alkyl of1 to 3 carbon atoms or together with U form an additional bond and R₂ isamine protecting group, an amino acid or a peptide of 2 to 4 amino acidswith the amine protected to obtain a compound of the formula ##STR11##in all isomeric forms or isomeric mixtures which is optionally isolatedand salified or subjected to the following reactions in any order:

a) if X' or Y' is COO-Alk₃ : decarboyxlation,

b) deprotection of the amino groups,

c) dealkylation of the hydroxyl groups,

d) amidation of the free amino groups with an amino acid or a peptide of2, 3 or 4 amino acids in which the amino group is protected, followed bydeprotection of this amino group,

e) esterificaiton or salification of the carboxy groups with a base, and

f) salification of the amino groups with an acid.

In a preferred mode of the process of the invention, the glutamic acidcompound of formula II is activated by formation of a mixed anhydridesuch as with isobutyl chloroformate or by the presence of a condensationagent such as dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole orbis(alkylamides) of sulfurous acid such as ##STR12## The decarboxylationis preferably effected with a concentrated inorganic acid such as 12Nhydrochloric acid. A direct decarboxylation may be effected directly bythe procedure of Krapcho or Keinan et al [J. Org. Chem., Vol. 51 (1986),p. 3615-3619].

The deprotection of the amino groups (formyl or BOC, for example) ispreferably carried out with a dilute mineral acid such as hydrochloricacid. The dealkylation of the carboxyl groups is preferably carried outby saponification with an inorganic base such as potassium hydroxideand, preferably sodium hydroxide. It can, if necessary, be performed intwo steps in the event of being incomplete. In the latter case, thesecond dealkylation is preferably carried out after the deprotection ofthe amino groups and/or the decarboxylation. The amidation of the freeamino groups is carried out, for example, using a functional derivativesuch as a halide of the amino acid or peptide, or in the presence of acondensation agent such as those mentioned above. The esterification ofthe carboxyl groups is carried out, for example, under the sameconditions as above (condensing agents).

The compounds of formula I may have a basic or acidic nature and if so,the addition salts thereof may be prepared by reaction with theappropriate base or acid in substantially stoichiometric form.

In a preferred embodiment of the process, the compound of formula I_(A)is subjected to the following reactions: dealkylation of the carboxylgroups, deprotection of the amino groups and decarboxylation, amidationor salification of the amino groups with an acid and/or esterificationor salification of the carboxyl groups with a base.

Some of the compounds of formula III are known and if they are notknown, they may be prepared by reacting a compound of the formula##STR13## wherein Alk₁, Alk₂, U, X', Y' and R₂ ' have the abovedefinitions with an alkanesulfonyl halide of 1 to 3 alkyl carbon atoms,preferably methane sulfonyl chloride, in the presence of a condensationagent such as pyridine and then with an alkali metal azide such assodium azide or diphenylphosphoryl azide to form a compound of theformula ##STR14## reducing the latter with triphenyl phosphine, forexample, followed by aqueous hydrolysis or by catalytic hydrogenation inthe presence of a Lindlar catalyst (poisoned palladium on charcoal) toform a compound of the formula ##STR15## wherein₂, X', Y', U, Alk₁ andAlk₂ have the above definitions and when X' and/or Y' are other thanhydrogen, reacting the latter with an agent to protect the amino groupto form a compound of the formula ##STR16## wherein R₂ ', Alk₁, Alk₂, U,X' and Y' have the above definitions and Rp is an amino protectivegroup, subjecting the latter to a decarboxylating agent to obtain thecorresponding compound with 1 or 2 asymetrical carbon atoms, separatingthe latter into the isomers or diastereoisomers such as bycrystallization or chromatography and then removing the amino protectivegroup to form the compound of formula III in the form of separateisomers or isometric mixtures.

The compounds of formulae II and IV are known.

The novel immunomodulatory compositions of the invention are comprisedof an immunomodulatorily effective amount of at least one compound offormula I and its non-toxic, pharmaceutically acceptable salts withacids and bases and an inert pharmaceutical carrier or excipient. Thecompositions may be in the form of tablets, dragees, gelatin capsules,granules, suppositories and injectable solutions or suspensions.

Examples of suitable excipients are talc, gum arabic, lactose, starch,magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatsof animal or vegetable origin, paraffinic derivatives, glycols, thevarious wetting, dispersing or emulsifying agents and preservatives.

The compositions of the invention have exceptional immuno modulatoryproperties, particularly by activation of human monocytes and productionof monokines such as TNF (tumor necrosis factor) and IL-1,interleukin-1.

They are useful in the treatment of autoimmune diseases, whetherconditions affecting organs nonspecifically (rheumatoid polyarthritis,lupus erythematosus, haemolytic anaemia, autoimmune leukopenia, and thelike) or specific diseases of organs (thyroiditis, Basedow's disease,Addison's disease, multiple sclerosis, pemphigus, haemorrhagicrectocolitis, some types of nephropathy, and the like). Compositions arealso useful in the treatment of haemopathies, cancer, AIDS, viral andmicrobial conditions, especially those which are chronic and recurrent(bronchitis, influenza and the like), diseases of the oral cavity, andthe like. They can constitute adjuvants of viral therapy, antibiotictherapy or anticancer chemotherapy.

The compositions also find use in the treatment of many secondary oracquired immune deficiency states observed during a wide variety ofconditions: deficiencies associated with metabolic disorders,deficiencies of iatrogenc origin (corticoids, ionizing radiation, etc.),deficiencies observed in major burns patients, and the like.

The novel method of the invention for inducing immunomodulatory activityin warm-blooded animals, including humans, comprises administering towarm-blooded animals an immunodulatorily effective amount of at leastone compound of formula I and its non-toxic, pharmaceutically acceptablesalts with acids and bases. The compounds may be administered orally,rectally or parenterally and the usual daily dose is 0.007 to 0.7 mg/kgdepending on the compound, condition treated and method ofadministration. For example, the compound of Example 1 and/or 8 may beorally administered at a dose 0.007 to 0.7 mg/kg for the treatment ofrheumatoid polyarthritis.

In the following examples there are described several preferredemobdiments to illustrate the invention. However, it is to be understoodthat the invention is not intended to be limited to the specificembodiments.

EXAMPLE 1 2-amino-6-(γ-D-glutamylamino)-4-methylene-heptanedioic acidSTEP A: Triethyl1-formylamino-3-methylene-5-[(N-trifluoroacetyl-0-methyl-γ-D-glutamyl)-amino-1,1,5-pentanetricarboxylate

A solution of 4 g of 1-methyl N-trifluoroacetyl-D-glutamate in 20 ml ofdimethoxyethane was added to a solution of 5.1 g of triethyl5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate in 280ml of dimethylethylamine and the mixture was cooled to 0° C. 3.2 g ofdicyclohexylcarbodiimide were added in small portions and the mixturewas stirred for 16 hours at 0° C. and filtered. The filtrate wasevaporated to dryness and the residue was taken up with colddimethoxyethane. The mixture was filtered and the filtrate was taken todryness. The residue was taken up in methylene chloride and the mixturewas washed with N hydrochloric acid, with saturated sodium bicarbonatesolution and then with water saturated with sodium chloride, dried andevaporated to dryness. The residue was purified by chromatography onsilica (eluant:ethyl acetate/cyclohexane 5:5) to obtain 5 g of theexpected product with a specific rotation [α]_(D) =+8° (c=1% inmethylene chloride).

STEP B: 2-amino-6-(γ-D-glutamylamino)-4-methylene-heptanedioic acidSaponification

10.9 ml of N sodium hydroxide were added dropwise over 5 minutes at 0°C. to a solution of 1.28 g of the product of Step A in 20 ml of ethanoland the mixture was allowed to return to room temperature and stirredfor 24 hours. It was neutralized with 0.9 ml of 12N hydrochloric acidand taken to dryness under reduced pressure.

Decarboxylation--deformylation

The residue was taken up in 10 ml of ethanol and 1 ml of 12Nhydrochloric acid was added and the mixture was stirred for 40 minutesat 80° C.

Saponification

The above product was chilled and 15.2 ml of 2N sodium hydroxide wereadded dropwise over 5 minutes. The mixture was stirred for 24 hours atroom temperature, brought to pH 6 using 12N hydrochloric acid and takento dryness.

Purification

The above product was taken up in a few ml of water and the pH wasadjusted to 6 with N sodium hydroxide. The mixture was dealkalinized onDowex 50 W×8 H⁺ resin (50-100 mesh) and elution was performed first withwater and then with N ammonia solution. The product was purified bychromatography on silica eluant:ethanol/ammonia solution 95:5, thenethanol/ammonia solution 8:2) and the eluant was evaporated to dryness.The residue was taken up in 200 ml of water and the mixture wasfiltered. The filtrate was lyophilized for 16 hours to obtain 400 mg ofthe expected product with a specific rotation [α]_(D) =-9.5°±1° (c=1% in4N hydrochloric acid).

A) Preparation of 1-methyl-N-trifluoroacetyl-D-glutamate Step 1:5-(1,1-dimethylethyl)-1-methyl-N-trifluoroacetyl-D-glutamate

4.1 ml of triethylamine and then, while cooling 4.1 ml oftrifluoroacetic anhydride were added dropwise to a solution of 5.5 g5-(1,1-dimethylethyl)-1-methyl-D-glutamate in 180 ml of methylenechloride. The mixture was then stirred for 2 hours at room temperatureand poured into 70 ml of ice-cold water. The resulting mixture wasextracted with methylene chloride and the extract was washed with salinesolution, dried and taken to dryness to obtain 7.1 g of the expectedproduct with a specific rotation of [α]_(D) =+5°±1° (c=1.3% in dioxane).

STEP 2: 1-methyl-N-trifluoroacetyl-D-glutamate

6.8 g of the product of Step 1 were dissolved in 50 ml of methylenechloride and 50 ml of trifluoroacetic acid were added. The mixture wasstirred for 1 hour and evaporated to dryness. The residue was taken upin ethyl acetate and the mixture was poured into water. The resultingmixture was extracted with ethyl acetate and the extract was washed withsaline solution and taken to dryness to obtain 5.5 g of the expectedproduct with specific rotation of [α]_(D) =+9°±2° (c=0.6% in dioxane).

B) Preparation of the starting triethyl5-amino-1-(formylamino)-3-methylene-1,5-pentanetricarboxylate STEP 1:Diethyl 2-(formylamino)-(2-methyl-2-propenyl)-propane-dioate

40 g of potassium carbonate, 0.380 g of 18-crown-6-crown ether catalystand 39.9 g of chloromethylpropene were added to a solution of 30 g ofethyl formamidomalonate in 300 ml of cyanomethyl and the mixture wasstirred at reflux for 3 hours and filtered. The filtrate was evaporatedto dryness and the residue was cooled to 0° C. to 5° C. and taken up in10 ml of isopropyl ether. The mixture was filtered, washed withisopropyl ether and dried under reduced pressure to obtain 25.2 g of theexpected product with a melting point of 72° C.

STEP 2: Triethyl1-(formylamino)-5-hydroxy-3-methylene-1,1,5-pentanetricarboxylate(mixture of isomers)

A solution of 26.5 g of ethyl glyoxylate in 180 ml of methylene chloridewas added dropwise over 10 minutes to a solution of 84 g of ferricchloride in 180 ml of methylene chloride and the mixture was stirred for1 hour and cooled to -20° C. A solution of 34 g of the product of Step Ain 180 ml of methylene chloride was added dropwise over 20 minutes, andthe mixture was stirred for 1 hour at -20° C. and was then poured into300 ml of ice-cold water. The resulting mixture was extracted withmethylene chloride and the extract was washed with 2N NCl and then withsaline solution, dried and evaporated to dryness under reduced pressureto obtain 56.5 g of the expected product (mixture of isomers) with amelting point of 68° C. after purification by chromatography on silica(eluant:ethyl acetate/cyclohexane 6:4)

STEP 3: Triethyl5-azido-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate

46 g of the product (mixture of isomers) of Step 2 were dissolved in 500ml of pyridine and the mixture was cooled to 0° C. 12 ml ofmethanesulfonyl chloride were added dropwise at 0° C. and the mixturewas stirred for 3 hours at room temperature and poured into 400 ml ofice-cold 4N hydrochloric acid and 200 ml of methylene chloride. Theresulting mixture was extracted with methylene chloride and the extractwas washed with 4N hydrochloric acid, with saturated sodium bicarbonatesolution and with saline solution, dried and evaporated to dryness underreduced pressure. The 56 g of residue obtained were dissolved in 250 mlof dimethylformamide and 9.98 g of sodium azide were added. The mixturewas stirred for 16 hours at room temperature and the solvent wasremoved. The residue was taken up in methylene chloride and the mixturewas washed with saturated sodium bicarbonate solution and then withsaline solution, dried and taken to dryness to obtain 59 g of expectedproduct (mixture of isomers) which was purified by chromatography onsilica (eluant:cyclohexane/ethyl acetate 8:2) to obtain 26 g of theexpected product.

STEP 4: Triethyl5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate

10.9 g of triphenylphosphine were added to a solution of 13 g of theproduct of Step 5 in 250 ml of tetrahydrofuran at approximately -5° C.and the mixture was stirred for 5 hours at room temperature. 8.5 ml ofwater were added and the mixture was stirred for 24 hours at roomtemperature. The tetrahydrofuran was evaporated off and the residue wastaken up in methylene chloride. The mixture was poured into ice-cold 2Nhydrochloric acid, followed by extraction with 2N hydrochloric acid,neutralization with sodium bicarbonate and extraction with methylenechloride. The extract was washed with saline solution, dried andevaporated to dryness to obtain 10 g of the expected product melting at50° C. after crystallization from isopropyl ether.

EXAMPLE 22-(L-Alanylamino)-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acidSTEP A: Triethyl1-amino-3-methylene-5-(N-trifluoroacetyl-0-methyl-γ-D-glutamyl)-amino]-1,1,5-pentanetricarboxylate

1.5 ml of 12N hydrochloric acid were added to a solution of 1.22 g ofproduct of Step A of Example 1 in 15 ml of ethanol and the mixture wasstirred for 40 minutes at 80° C. The solution was removed under reducedpressure and the residue was taken up in 10 ml of water. The mixture wasneutralized with sodium bicarbonate and extracted with methylenechloride. The extract was washed with saline solution, dried and takento dryness. The residue was purified by chromatography on silica(eluant:ethyl acetate/cyclohexane 8:2) to obtain 0.850 g of the expectedproduct.

STEP B: Triethyl1-(N-[(1,1-dimethylethoxy)-carbonyl]-L-alanyl-amino-3-methylene-5-[(N-trifluoroacetyl-0-methyl-γ-D-glutamyl)-amino]-1,1,5-pentanetricarboxylate

0.310 g of BOC-L-alanine were dissolved in 50 ml of tetrahydrofuran and0.43 ml of triethylamine were added. The mixture was cooled toapproximately 5° C. and 0.23 ml of isobutyl chloro formate were added.The mixture was stirred for 30 minutes at approximately +5° C. and asolution of 0.800 g of the product of Step A in 10 ml of tetrahydrofuranwas added dropwise over 5 minutes. The mixture was allowed to return toroom temperature and was stirred for 16 hours. The tetrahydrofuran wasremoved and the residue was taken up in a water/methylene chloridemixture. The mixture was extracted with methylene chloride and theextract was washed with saline solution, dried and taken to dryness. Theresidue was purified by chromatography on silica (eluant:ethylacetate/cyclohexane 5:5) to obtain the expected product with a specificrotation of [α]_(D) =12°±1° (c=1% in methylene chloride).

STEP C: 2-(L-alanylamino)-6-(γ-D-glutamylamino)-4-methyleneheptanedioicacid

Using the procedure of Step B of Example 1, 700 mg of the product ofStep B were reacted. Saponification, deprotection of the BOC-protectedamine, saponification and purification were performed to obtain 170 mgof the expected product with a specific rotation of [α]_(D) =-13°±1°(c=1% in 4N HCl).

EXAMPLE 32-Amino-4-methylene-6-[N-(1-oxo-octadecyl)-L-alanyl-γ-D-glutamyl-amino]-heptanedioicacid

Using the procedure of Example 1,1-methyl-N-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamate was used in placeof the corresponding N-trifluoroacetyl derivative obtained in thepreparation A of Example 1, to obtain the expected product with aspecific rotation of [α]_(D) =-25° (c=0.6% in water).

Preparation of 1-methyl N-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamateSTEP 1: Methyl N-(1-oxo-octadecyl)-L-alaninate

24.9 ml of triethylamine were added to a solution of 10 g of L-alaninemethyl ester hydrochloride in 250 ml of methylene chloride, followed bydropwise addition over 30 minutes of a solution of 21.7 g of stearylchloride in 50 ml of methylene chloride. The mixture was stirred for 150minutes and filtered and the filtrate was washed with 2N hydrochloricacid, then with saline solution, dried and taken to dryness underreduced pressure. The residue was made into a paste with isopropylether, vacuum filtered and crystallized in methanol to obtain 22 g ofthe expected product with melting point of 84° C. and with a specificrotation of [α]_(D) =-15° (c=1% pyridine).

STEP 2: N-(1-oxo-octadecyl)-L-alanine

A suspension of 21 g of above product in 500 ml of methanol was cooledat 0° C. and 62 ml of N potassium hydroxide were added dropwise. Themixture was stirred for 16 hours at room temperature and 500 ml ofmethanol were added. The mixture was stirred for 4 hours, cooled to 0°C., brought to a pH of 3 using 2N hydrochloric acid and evaporated. Theresidue was taken up in methylene chloride and the mixture was washedwith saline solution, dried and taken to dryness under reduced pressure.The residue was made into a paste with isopropyl ether vacuum filteredand crystallized from methanol to obtain 11.8 g of the expected productmelting at 102° C. and having a specific rotation of [α]_(D) =-18°(c=0.9% in pyridine).

STEP 3: 5-(1,1-dimethylethyl)-1-methylN-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamate

A suspension of 1.77 g of N-(1-oxo-octadecyl)-L-alanine and 1.08 g of5-(1,1-dimethylethyl)-1-methyl D-glutamate in 170 ml of dimethoxyethanewas cooled to 0° C. and a solution of 1.23 g ofN,N-dicyclohexylcarbodiimide in 5 ml of dimethoxyethane was addeddropwise over 10 minutes. The mixture was stirred for 3 hours at roomtemperature, cooled and filtrated. The filtrate was evaporated todryness and the residue was taken up in 250 ml of ether in the heatedstate. The mixture was filtered and the filtrate was evaporated todryness under reduced pressure. The residue was made into a paste inisopropyl ether and vacuum filtered to obtain 2.4 g of the expectedproduct melting at ≃ 100° C. after crystallization in methanol andhaving a specific rotation of [α]_(D) =-32°±1° (c=1% in CH₂ Cl₂).

STEP 4: 1-Methyl N-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamate

31 ml of trifluoroacetic acid were added dropwise to a solution of 11.4g of the product of Step 3 in 150 ml of methylene chloride and themixture was left for 16 hours with stirring and then evaporated todryness under reduced pressure. The residue was taken up in methylenechloride and the mixture was washed with saline solution, dried andfiltered. The filtrate was evaporated to dryness under reduced pressureand the residue was then crystallized from ethanol to obtain 6.8 g ofthe expected product melting at ≃ 110° C. and having a specific rotationof [α]_(D) =-16°±1° (c=0.9% in pyridine).

EXAMPLE 4 2-Amino-6-(γ-D-glutamylamino)-3-heptenedioic acid

Using the procedure of Example 1, triethyl5-amino-1-formylamino-2-pentene-1,1,5-tricarboxylate was reacted toobtain the expected product.

Preparation of triethyl5-amino-1-formylamino-2-pentene-1,1,5-tricarboxylate

Using the procedure for the preparation of triethyl5-amino-1-(formylamino)-3-methylene-1,1,5-pentanetricarboxylate,chloro-methylpropene replaced the allyl bromide to obtain the expectedproduct in the form of an oil with an Rf=0.4 in ethyl acetate/ethanol4:1.

EXAMPLE 52-Glycylamino-6-[N-(1-oxo-dodecyl)-L-alanyl-γ-D-glutamyl]-amino-2-heptenedioicacid

Using the procedure of Example 1, 1-methylN-[N-(1-oxo-dodecyl)-L-alanyl]-D-glutamate [prepared like 1-methylN-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamate in Example 3 by replacingstearyl by lauryl (dodecyl)] was reacted with7-ethyl-1-methyl-6-amino-2-[(N-formylglycyl)-amino]-3-heptenedioate. Inthis case, deformylation with hydrochloric acid was not necessary andthe neutralization and the desalification were carried outsimultaneously using a H⁺ resin (Amberlyst 15). The expected product,which became resinous at 150° C., was obtained and had a specificrotation of [α]_(D) =-14° (c=0.8% in water).

EXAMPLE 6 Mixture of (2D, 6L)- and (2L,6D)-2-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid STEP A:Mixture of (2D, 6L)- and (2L,6D)-2-formylamino-4-methylene-6-[N-(trifluoroacetyl)-γ-D-glutamylamino]-heptanedioicacid

1.3 g of dicyclohexylcarbodiimide were added at 0° C. to a solutioncontaining 1.5 g of the mixture of diethyl (2D,6L)- and (2L,6D)-2-formamido-4-methylene-6-amino-1,7-heptanedioate and 1.48 g ofD-glutamic acid in 150 ml of dimethoxyethane, and the resulting mixturewas stirred for 16 hours while the temperature was maintained at 0° C.The precipitate was filtered off and the filtrate was concentrated todryness under reduced pressure. The 10.5 g of crude product werepurified by chromatography on silica (eluant:cyclohexane/ethyl acetate2:8) to obtain the expected product with a specific rotation of [α]_(D)=-14° (c=0.8% in water).

STEP B: Mixture of (2D, 6)- jand (2L,6D)-2-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid

5 ml of 12N hydrochloric acid were added to 1.9 g of the product of StepA dissolved in 100 ml of ethanol, and the mixture was stirred for 30minutes. The solvent was removed under reduced pressure and the residuewas taken up in 50 ml of ethanol. The pH was adjusted to 7 with 0.1Nsodium hydroxide solution and the mixture was cooled to 0° C. and 16 mlof N sodium hydroxide were added dropwise. The reaction medium wasallowed to return to room temperature, stirred for 16 hours andneutralized with N hydrochloric acid. The solvents were removed underreduced pressure and the residue was chromatographed on silica(eluant:ethanol/ammonia solution 95:5, 90:10 and then 80:20). The resinobtained was taken up in water and the mixture was filtered and thefiltrate was lyophilized to obtain 0.92 g of expected product with aspecific rotation of [α]_(D) =-13°±1° (c=1% in 3N HCl).

EXAMPLE 7 Mixture of (2D, 6D)- and (2L,6L)-2-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acids STEP A:Mixture of (2D, 6D)- and (2L,6L)-2-formylamino-4-methylene-6-[N-(trifluoroacetyl)-γ-D-glutamylamino]-heptanedioicacids

Using the procedure of Step A of Example 6, a mixture of diethyl (2D,6D)- and (2L, 6L)-2-formamido-4-methylene-6-amino-1,7-heptanedioates wasreacted to obtain the expected product.

STEP B: Mixture of (2D,6D)- and(2L,6L)-2-amino-6-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioicacids

Using the procedure of Step B of Example 6, the product of Step A wasreacted to obtain the expected product having a specific rotation of[α]_(D) =-12.5°±1° (c=[1%], 3N HCl).

The mixture of diethyl (2D,6D)- and(2L,6L)-2-formamido-4-methylene-6-amino-1,7-heptanedioates used at thebeginning of Example 7 was prepared as described in the preparation ofthe Example (Step C), starting with the mixture of diethyl (2D,6D)-and(2L,6L)-2-formamido-4-methylene-6-tert-butoxycarbonylamino-1,7-heptanedioates(isomer I) obtained in Step B of this preparation.

Preparation of 7-ethyl 1-methyl6-amino-2-[(N-formylglycyl)-amino]-3-heptanedioate

Using the procedure of preparation B of Example 1,diethyl-(formylamino)-(2-methyl-2-propenyl)-propanedioate in Step 2 wasreplaced by methyl 2-[(N-formylglycyl)-amino]-2-(2-propenyl)-ethanoateto obtain the expected product with an Rf=0.2 in a methylenechloride/methanol (9:1) mixture.

Comment

The reduction of the azide to amine (Step 4 of preparation B ofExample 1) was carried out by hydrogenation in the presence of palladiumon calcium carbonate and of quinoline, instead of triphenyl phosphine.

Preparation of the mixture of diethyl (2D, 6L)- and (2L,6D)-2-formamido-4-methylene-6-amino-1,7-heptanedioates STEP A: Triethyl1-formamido-3-methylene-5-(tert-butoxycarbonylamino)-1,1,5-pentanetricarboxylate

13.6 g of di-tert-butyl pyrocarbonate dissolved in 25 ml of methylenechloride were added at room temperature to 19.5 g of triethyl1-formamido-3-methylene-5-amino-1,1,5-pentanetricarboxylate dissolved in75 ml of methylene chloride, and the mixture was maintained withstirring at room temperature for 36 hours. The reaction medium wasdiluted with 150 ml of methylene chloride and the organic phase waswashed with water and dried. The solvents were removed under reducedpressure and the mixture was left to crystallize at +4° C. to obtain25.8 g of expected product melting at 68° C.

STEP B: Mixture of diethyl (2D,6D)- and(2L,6L)-2-formamido-4-methylene-6-(tert-butoxycarbonylamino)-1,7-heptanedioatesand mixture of (2D,6L) and (2L,6D) isomers

2.21 g of cesium carbonate and 9.42 g of p-aminophenol were added to17.25 g of product of Step A dissolved in 250 ml of dimethylformamide,and the mixture was heated for 3 hours to 85° C. The mixture was allowedto room temperature and was filtered. The solvents were removed underreduced pressure and the residue was taken up in 300 ml of methylenechloride. The organic phase was washed with 0.5N hydrochloric acid andthen with aqueous sodium bicarbonate solution and dried. The solvent wasremoved under reduced pressure and the residue was chromatographed on asilica column (eluant:cyclohexane/ethyl acetate 6:4) to obtain 4.70 g ofisomer I (DD,LL isomer), 3.84 g of isomer II (DL, LD isomer) and 2.80 gof a mixture of the 2 isomers.

STEP C: Mixture of diethyl (2D,6L)- and(2L,6D)-2-formamido-4-methylene-6-amino-1,7-heptanedioates

25 ml of 2N hydrochloric acid in ether were added to 3 g of product IIof Step B (DL,LD isomer) dissolved in 50 ml of methylene chloride. Themixture was stirred at room temperature for 30 minutes and concentratedunder an inert atmosphere. The amine hydrochloride was taken up inmethylene chloride to which triethylamine had been added, and themixture was then evaporated to dryness under reduced pressure. Afterchromatography on silica (eluant:ethyl acetate/cyclohexane 7:3), 1.86 gof expected product were recovered.

EXAMPLE 82-Amino-4-methylene-6-[N-1-oxo-octadecyl)-L-alanyl)-γ-D-glutamyl-amino]-heptanedioicacid and its tri sodium salt STEP A:Diethyl-2-formamido-(-2-methyl-2-propenyl)-propanedioate

A solution of 30 g of ethyl formamido-malonate in 300 ml of acetonitrilewas added to a mixture of 40 g of potassium carbonate, 0.380 g of 18crown 6 ether catalyst and 39.9 g of chloromethylpropane and the mixturewas refluxed for 3 hours and then filtered. The filtrate was dried andcooled to 0° to 5° C. The precipitate was taken up in isopropyl etherand filtered. The product was washed with isopropyl ether and was driedunder reduced pressure to obtain 25.2 g of the desired product meltingat 72° C.

STEP B: Triethyl1-formamido-5-hydroxy-3-methylene-1,1,5-pentane-tridicarboxylate

A solution of 26.5 g of ethyl glycoxylate in 180 ml of methylenechloride was added dropwise over 10 minutes to a solution of 84 g offerric chloride in 180 ml of methylene chloride and the mixture wasstirred for one hour, then cooled to -20° C. A solution of 34 g of theproduct of Step A was added dropwise over 20 minutes to the mixturewhich was then stirred at -20° C. for one hour and was poured into 300ml of ice water. The mixture was extracted with methylene chloride andthe organic phase was washed with 2N hydrochloric acid, then aqueoussodium chloride solution, dried and evaporated to dryness under reducedpressure to obtain 56.5 g of a mixture of isomers of the expectedproduct melting at 68° C. after chromatography over silica (6-4 ethylacetate/cyclohexane).

STEP C: Triethyl5-azido-1-formamido-3-methylene-1,1,5-pentane-tricarboxylate

12 ml of methanesulfonyl chloride were added dropwise at 0° C. to asolution of 46 g of the mixture of Step B in 500 ml of pyridine andafter stirring for 3 hours at room temperature, the mixture was pouredinto 400 ml of iced 4N hydrochloric acid and 200 ml of methylenechloride. The mixture was extracted with methylene chloride and theorganic phase was washed with 4N hydrochloric acid, then with saturatedsodium bicarbonte solution, then with aqueous sodium chloride solution,and evaporated to dryness under reduced pressure. The 56 g of residuewere dissolved in 250 ml of dimethylformamide and after the addition of9.98 g of sodium azide, the mixture was stirred at room temperature for16 hours and evaporated to dryness. The residue was taken up inmethylene chloride and the solution was washed with saturated sodiumbicarbonate solution, then with aqueous sodium chloride solution, driedand evaporated to dryness to obtain 59 g of the desired mixture ofisomers. Chromatography over silica and elution with a 8-2 cyclohexanemethyl acetate mixture yielded 26 g of the said product.

STEP D: Triethyl 5-amino-1-formamido-3-methylene-1,1,5-pentanetricarboxylate

10.9 g of triphenylphosphine were added to a solution of 13 g of theproduct of Step C in 250 ml of tetrahydrofuran at about -5° C. and afterstirring for 5 hours at room temperature, 8.5 ml of water were added.The mixture was stirred at room temperature for 24 hours and wasevaporated to dryness. The residue was taken up in methylene chlorideand the solution was added to iced 2N hydrochloric acid. The mixture wasextracted with 2N hydrochloric acid and the extract was neutralized withsodium bicarbonate. The mixture was extracted with methylene chlorideand the organic phase was washed with sodium chloride solution, driedand evaporated to dryness to obtain 10 g of the expected product meltingat <50° C. after crystallization from isopropyl ether.

STEP E: Triethyl5-(tert.-butoxycarbonylamino)-1-formamido-3-methylene-1,1,5-pentane-tricarboxylate

A solution of 15.65 g of ditert.-butyl pyrocarbonate (Bo C₂ O) in 25 mlof dichloromethane was added to a solution of 19.5 g of the product ofStep D in 75 ml of dichloromethane and the mixture was stirred for 36hours in a water bath at room temperature. The mixture was diluted with150 ml of dichloromethane and was washed with water, filtered and driedto obtain 25.8 mg of the expected product melting at 68° C.

STEP F: Diethyl6-(tert-butoxycarbonylamino)-2-formamido-4-methylene-heptanedioate

2.21 g of cesium carbonate and 9.42 g of p-amino-thiophenol were addedall at once to a solution of 17.25 g of the product of Step E in 250 mlof dimethylformamide and the mixture was heated at 85° C. for 3 hoursand was then returned to room temperature. The solution was filtered andthe filtrate was evaporated to dryness under reduced pressure. The oilresidue was taken up in 300 ml of chloromethane and the solution waswashed with hydrochloric acid and then with a saturated aqueous sodiumbicarbonate solution. The oil was purified by HPLC and elution with a6-4 cyclohexane-ethyl acetate mixture yielded the expected product.

STEP G: Diethyl 2-amino-6-formamido-4-methylene-heptanedioate

100 ml of a solution of 2.6N hydrochloric acid in anhydrous ether wereadded to a solution of 6.4 g of the product of Step F in 200 ml ofmethylene chloride and after stirring at room temperature for one hour,nitrogen was bubbled therethrough The mixture was evaporated to drynessunder reduced pressure at 30° C. and the residue was taken up inmethylene chloride. The mixture was neutralized and evaporated todryness to obtain 4.6 g of raw product. The latter was chromatographedover silica and eluted with a 9-1 ethyl acetate-ethanol mixture toobtain the desired product with an Rf=0.06.

STEP H: Diethyl2-formamido-4-methylene-6-[[0-methyl-N-[N-(1-oxo-octadecyl)-L-alanyl]-.gamma.-D-glutamyl]-amino]-heptanedioate

0.58 ml of triethylamine and 0.3 ml of isobutyl chloroformate were addedat 14°±1° C. to a solution of 0.992 g of methylN-[N-(1-oxo-octadecyl)-2-alanyl]-D-glutamate acid, 50 ml of dioxane and50 ml of tetrahydrofuran and after stirring at 14° C. for 35 minutes, asolution of 0.572 g of the product of Step G in 10 ml of tetrahydrofuranwas added. The mixture was stirred at room temperature for 2 hours andwas evaporated to dryness at reduced pressure at 30° C. The residue waschromatrographed over silica and eluted with a 2.5-7.5 cyclohexane-ethylacetate mixture to obtain 480.6 mg of the desired diastereoisomer A and476 mg of the desired diastereoisomer B.

STEP I: Diethyl 2-amino 6-[[O-ethylN-[N-(1-oxo-octadecyl)-L-alanyl]-γ-D-glutanyl]amino]4-methyleneheptanedioate

403 mg of the product of step H (isomer B) in ethanol were stirred for45 minutes at 80° C. with 0,41 ml of concentrated hydrochloric aciddiluted with 4,1 ml of ethanol. The mixture was then cooled and 2 ml ofwater were added. The mixture was neutralized with sodium bicarbonateand extracted with methylene chloride dried and evaporated to drynessunder reduced pressure. The residue was chromatographed to obtain thedesired product which is used as it is for the next step.

STEP J:2-amino-4-methylene-6-[[N-[N-(1-oxo-octadecyl)-L-alanyl]-γ-D-glutamyl]-amino]-heptanedioicacid and its tri sodium salt

0.43 ml of sodium hydroxide were added at 0° C. to a solution of 107 mgof the product of Step I in 5 ml of ethanol and the mixture was stirredovernight at room temperature and was evaporated to dryness to obtain117 mg of product which was added to 10 of apyrogen water. The mixturewas filtered and lyophylized to obtain 50 mg of the desired product witha specific rotation of [α]_(D) =-25°±2° (c=1% in H₂ O).

PREPARATION STEP A: Methyl N-(1-oxo-octadecyl)-L-alaninate

24.9 ml of triethylamine were added to a solution of 10 g of methylL-alaninate hydrochloride in 250 ml of methylene chloride and then asolution of 21.7 g of stearyl chloride in 50 ml of methylene chloridewas added dropwise over 30 minutes. The mixture was stirred for 21/2hours and filtered. The filtrate was washed and with 2N hydrochloricacid, then with aqueous sodium chloride, dried and evaporated to drynessunder reduced pressure. The residue was empasted with isopropyl ether,vacuum filtered and crystallized from methanol to obtain 22 g of thedesired product melting at 84° C. and having a specific rotation of[α]_(D) =-15° (c=1% in pyridine)

STEP B: N-(1-oxo-octadecyl)-L-alaninate

62 ml of N potassium hydroxide solution were added dropwise at 0° C. toa suspension of 21 g of the product of Step A in 500 ml of methanol andafter stirring at room temperature for 16 hours, 500 ml of methanol wereadded. The mixture was stirred for 4 hours, cooled to 0° C. and the pHwas adjusted to 3 with 2N hydrochloric acid. The mixture was evaporatedto dryness and the residue was taken up in methylene chloride. Thesolution was washed with aqueous sodium chloride, dried and evaporatedto dryness under reduced pressure. The residue was empasted withisopropyl ether, vacuum filtered and crystallized from methanol toobtain 11.8 g of the desired product melting at 102° C. and having aspecific rotation of [α]_(D) =-18° (c=0.9% in pyridine).

STEP C: 1-methyl N-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamate

A solution of 1.23 g of N,N-dicyclohexylcarbodiimide in 5 ml ofdimethoxyethane was added dropwise over 10 minutes at 0° C. to asuspension of 1.77 g of the product of Step B and 1.08 of1,1-dimethylethyl D-glutamate and after stirring at room temperature for3 hours, the mixture was cooled and filtered and evaporated to dryness.The residue was taken up in 250 ml of hot ether and the solution wasfiltered and empasted to dryness under reduced pressure. The residue wasevaporated with isopropyl ether, and vacuum filtered to obtain 2.4 g ofthe desired product melting at 100° C. after crystallization frommethanol and a specific rotation of [α]_(D) =-32°±1° (c=1% in CH₂ Cl₂).

STEP D: 1-methyl N-[N-(1-oxo-octadecyl)-L-alanyl]-D-glutamate

31 ml of trifluoroacetic acid were added dropwise to a solution of 11.4g of the product of Step C in 150 ml of methylene chloride and afterstirring for 16 hours, the mixture was evaporated to dryness underreduced pressure. The residue was taken up in methylene chloride and thesolution was washed with sodium chloride solution, dried, filtered andevaporated to dryness under reduced pressure. The product wascrystallized from ethanol to obtain 6.8 g of the exposed product meltingat 110° C. and having a specific rotation of [α]_(D) =-16°±1° (c=0.9% inpyridine)

EXAMPLE 192-Amino-4-methylene-6-[[N-(1-oxo-octadecyl)-γ-D-glutamyl]amino]-heptanedioicacid STEP A: Diethyl2-formamido-4-methylene-6-[[O-methyl-N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioate

0.62 ml of triethylamine and 0.3 ml of isobutyl chloroformate were addedat 14° C. over 5 minutes to a solution of 615 mg of1-methyl-N-(1-oxo-octadecyl)-glutamate in a 1-1 mixture of THF anddioxane and after stirring the suspension at 14° C. for 35 minutes, asolution of 915 mg of the product of Step G of Example 8 in 10 ml oftetrahydrofuran was added. The mixture was stirred at room temperaturefor 2 hours and was then added to ice and was extracted with methylenechloride. The organic phase was washed, dried, filtered and evaporatedto dryness at 30° C. to obtain 2.1 g of a mixture of diastereoisomers Aand B. The mixture was chromatographed over silica and eluted with a2.5-7.5 cyclohexane-ethyl acetate mixture to obtain 588 mg ofdiastereoisomer A of the expected compound and 580 mg of diastereoisomerB.

STEP B: Diethyl2-amino-4-methylene-6[[O-methyl-N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioate

0.5 ml of concentrated hydrochloric acid were added to a solution of 470mg of the diastereoisomer A of Step A in 4.7 ml of ethanol and themixture was heated to 80° C. for 45 minutes. 20 ml of water were addedand the pH was adjusted to 7. The mixture was extracted with methylenechloride and the organic phase was dried, filtered and evaporated todryness to obtain 500 mg of product which was purified by chromatographyover silica to obtain 251 mg of diastereoisomer A of the expectedproduct.

NMR Spectrum (CDCl₃.250 MHz)

Peaks at 3.64 (2-hydrogen of principal chain) and at 4.99 (hydrogen of4-methylene)

Using the same procedure, 580 mg of the diastereoisomer B of Step A werereacted to obtain 243 mg of the expected diastereoisomer B.

NMR Spectrum (CDCl₃ --250 MHz)

Peaks at 1.65 (hydrogen of 2-substituted amino); at 3.61 (2-hydrogen ofprincipal chain; at 4.94-4.98 (hydrogen of 4-methylene).

STEP C:2-Amino-4-methylene-6-[[N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioicacid (A and B diastereoisomers)

1.02 ml of a solution of 0.00102 M of sodium hydroxide were added at 0°C. to a solution of 233 mg of the A isomer product of Step B in ethanoland after stirring overnight at room temperature, the mixture wasevaporated to dryness under reduced pressure. The residue was dissolvedin 15 ml of water and the solution was filtered and lyophilized toobtain 133 mg of the expected diastereoisomer A with a specific rotationof [α]_(D) =-9°±2° (c=0.7% in H₂ O).

NMR Spectrum (D₂ O)

Peaks at 4.16 and 4.33 (6-hydrogen and hydrogen of CH of γ-D-glutyamyl);at 3.41 (2-hydrogen); at 0.89 (18-hydrogen of octadecyl).

Using the same procedure, 234 mg of the B isomer product of Step B werereacted to obtain 194.4 mg of the expected B isomer product with aspecific rotation of [α]_(D) =-8.5°±2° (c=0.7% in H₂ O).

NMR Spectrum (D₂ O)

Peaks at 4.15 and 4.32 (6-hydrogen and hydrogen of --CH-- ofγ-D-glutamyl); at 3.46 (2-hydrogen); at 0.88 (18-hydrogen of octadecyl).

The starting (1) methyl N-(1-oxo-octadecyl) acid glutamate used in StepA was prepared by the following reactions ##STR17##

EXAMPLE 10

Tablets were prepared containing 50 mg of2-amino-6-(γ-D-glutamylamino)-4-methylene-heptanedioic acid andsufficient excipient of lactose, starch, talc, magnesium stearate for afinal weight of 100 mg.

EXAMPLE 11

Tablets were prepared containing 50 mg of2-(L-alanylamino)-6-(γ-D-glutamylamino)-4-methylene-heptanedioic acidand sufficient excipient of lactose, starch, talc and magnesium stearatefor a final weight of 100 mg.

EXAMPLE 12

Tablets were prepared containing 50 mg of the product of example 8 andsufficient excipient of lactose starch, talc, magnesium stearate for afinal weight of 250 mg.

PHARMACOLOGICAL STUDY Stimulation of monocytic cells with animmunostimulant

The mononuclear cells of the circulating blood of normal donors wereseparated by the classical technique described by Boyum using a Ficollgradient. After being washed, the mononuclear cells were incubated at37° C. for 1 hour in the proportion of 5×10⁶ monocytes (NSE⁺ cells) perml of culture medium, 5 ml per culture bottle. The culture medium usedin this experiment was composed of RPMI 1640 to which antibiotics andHEPES buffer had been added. After one hour, the non-adherent cells wereremoved by washing the bottles with medium previously brought to 37° C.,and the adherent cells, composed essentially of monocytes (>90%), werecultured again in the presence of different amounts of test products ina PBS medium (Dulbecco) without Ca²⁺ or Mg²⁺. Culturing was continuedfor 24 or 48 hours and the cell supernatants were then withdrawn,centrifuged, aliquoted and stored either at -80° C. or at -20° C. Theculture supernatants were replaced in the bottles by the same amount ofpyrogen-free distilled water to lyse the cells. The lysate wasrecovered, aliquoted and also stored at -20° C. The followingexperiments were carried out in the presence or absence of interferon-(10³ U/ml) at a dose at which IFN-γ alone was inactive.

Tests for the presence in the supernants of monokines (interleukin-1 andtumor necrosis factor) on the basis of their biological activity.

Interelukin-1 (IL-1) Test

This test was first described by Gery et al in 1972 [Gery et al, 1972Potentiation of the T Lymphocyte response to mitogens. II. The CellularSource of Potentiating Mediator(s). J. Exp. Med., 136-143]. It is basedon the co-mitogenic action of IL-1 in the presence of an antigen(mimicked by phytohaemagglutinin in the test) on mouse thymocytes.1.5×10⁶ thymocytes of C₃ H/HeJ mice (supplied by C.S.E.A.L. of Orleans)were cultured for 3 days in the presence of different dilutions of cellsupernatants and lysates likely to contain IL-1 activity, and WellcomePHA-P (1 μg/ml), in culture plates having 96 flat-bottomed wells, in afinal volume of 200 μl of medium composed of RPMI 1640 containing, inaddition to antibiotics (penicillin 1 μ/ml streptomycin 1000 μ/ml, 1 mMHEPES buffer, 2 mM glutamine, 5% calf serum and 5×10⁻⁵ M2-mercaptoethanol). After 68 hours of culturing, 1 μCi of tritiatedthymidien was added to each well ([methyl³ H]-thymidine, CEA Saclay,TMM79A, specific activity 1 μCi/mM), and the radioactivity incorporatedby the cells was measured after the cultures had been filtered on aSkaton type semi-automatic collecting apparatus and the filters countedin a scintillation counter (LKB). The results were expressed as thedifference between the pulses per minute incorporated by the cultures inthe presence of supernatants and the pulses per minute incorporated bythe control cultures.

Tumor Necrosis Factor (TNF) Test

TNF activity was demonstrated by the toxicity of this factor on L-929target cells (sub-clone α). The technique was sensitized by addingactinomycin D to the test. The L cells were distributed in theproportion of 2×10⁴ cells per well of a flat-bottomed microplate in 100μl of RPMI 1640 medium enriched with 5% calf serum, glutamine, HEPESbuffer and antibiotics. After 24 hours, different dilutions of the testsupernatants were added in a volume of 100 μl, as well as a dose ofactinomycin D of 1 μg/ml. After 24 hours of culturing, the number ofunlysed viable cells was measured by staining the plates with crystalviolet and measuring the optical density of different wells on amultiscan reader.

RESULTS

The products of Examples 1, 2, 8 and 9 stimulated monocytes and theirIL-1 and TNF production. In addition, there was synergy between theproducts of Examples 1, 2, 8 and 9 and interferon-γ and or LPS.

Stimulation of the lymphocyte cells by an immuno stimulant Test forlymphoblast transformation

The mono-nuclear cells of circulating blood are put in culture at a rateof 2.10⁶ cells/ml in flat bottom micro-plates with 96 wells (Nunc) in100 μl of RPMI 1640 medium enriched with 10% of foetal calf serum(Hyclone), antibiotics, glutamine and Hepes. The products to be testedare added into the wells at a rate of 100 μl to the final dilutions of1.10 or 100 μg/ml either alone or in presence of phytohemaglutininePHA-P (Welcome labs) at 1 and/or 10 μg/ml. The plates are put at atemperature of 37° C. in an incubator with CO₂ in an atmospheresaturated with humidity. At the end of 2 and 5 days, the cultures arestopped, an incorporation of tritiated thymidine is carried out in theconditions described above in the detection test for Interleukin-1. Theresults are given in impulsions per minute.

Summarizing, the products of the chemical examples have been shown to beneither cytotoxic for the lymphocytes in culture nor mitogenetic forthese same cells. On the other hand, added at the same time as oneproduct known to be non-specific T mitogenetic such as PHA, they havebeen shown to be capable of partly inhibiting (especially at 100 μg/ml)the effects of this product. These results could be reproduced on allthe donors tested (between 3 and 30 per product).

Production of immuno-globulins

The same cells were cultivated for a week in the presence of differentproducts to be tested at a dose of 0.1, 1 and 10 μg/ml alone or in thepresence of two doses of the bacteria Staphylococcus aureus Cowan (SAC)(10⁻³ and 10⁻⁴), bacteria known for stimulating the production ofimmuno-globulins by the human B lymphocytes in a non-specific manner.The supernatants are collected at the end of the culture, and theircontent of immuno-globulin is evaluated by standard techniques ofdetection by the ELISA method using a human anti-immunoglobulinanti-body available commercially. The results are given in ng ofimmuno-globulin per ml of supernatant.

The experiments showed us that the products used on their own are notcapable of causing stimulation of lymphocytes of the B type (neithertheir proliferation, nor their differentiation in plasmocytes) but inthe presence of SAC the product from Example 8 is capable of increasingthe production of immuno-globulins. On the other hand, the product ofthe example does not give the same result (see figure).

In vivo activity Activation of the macrophages

The ability to activate the cells of the monocyte macrophage compartmenthas been confirmed in vivo in mice. After an injection byintra-peritoneal route of different doses of the products to be tested,the peritoneal cells of the mice BALB/c were collected; the quantity ofthe cells found had not increased and they were essentially macrophages,which shows that these products are not chemotactic at least in theconditions used. The macrophages have the particularity of being able torespond better to a membranal stimulation which "mimikes" aphagocytosis; in effect, these cells do not spontaneously producesuperoxide anions or oxygenated water, but this oxidative metabolism isprepared and is released on the phagocyte signal. The experiments werecarried out with different products. The oxidative metabolism is madeevident by a chemiluminescence technique in the presence of luminol,using a Berthold LB950 chemiluminometer.

The kinetics of the reaction shows that the phenomenon of activationlasts from 24 to 48 hours following the single injection of the product.

Various modifications of the products and processes of the invention maybe made without departing from the spirit or scope thereof and it shouldbe understood that the invention is intended to be limited only asdefined in the appended claims.

What we claim is:
 1. A compound selected from the group consisting ofall isomeric forms and mixtures of isomers of glutamic acid compounds ofthe formula ##STR18## wherein the glutamic acid is of D- or L-configuration, R₁ is selected from the group consisting of hydrogen,alkyl of 1 to 5 carbon atoms, an amino acid, a peptide of 2 to 4 aminoacids and an amino acid or a peptide of 2 to 4 amino acids in which theamine is amidified with an optionally unsaturated aliphatic carboxylicacid of 6 to 24 carbon atoms or R₁ is selected from the group consistingof a residue of a C₆ -C₂₄ optionally unsaturated aliphatic acid, R₅ isselected from the group consisting of hydrogen and an alkyl of 1 to 5carbon atoms, R₃ is selected from the group consisting of hydroxy,alkoxy of 1 to 5 carbon atoms and an amino acid with the amineoptionally substituted with alkyl of 1 to 5 carbon atoms, z is ##STR19##R₂ is selected from the group consisting of hydrogen, an amino acid anda peptide of 2 to 4 amino acids, R₄ is selected from the groupconsisting of hydroxyl, alkoxy of 1 to 5 carbon atoms and an amino acidoptionally substituted on the amine with alkyl of 1 to 5 carbon atoms, Uis selected from the group consisting of ##STR20## --CH═CH--CH₂ -- (E orZ isomer), --CH₂ --CH═CH-- (E or Z isomer) and ##STR21## or U and Ytogether are ═CH--CH₂ --CH₂ -- (E or Z isomer) and X is hydrogen or Uand X together are ═CH--CH₂ --CH₂ --(E or Z isomer) and Y is hydrogenand their salts with non-toxic, pharmaceutically acceptable acid orbases.
 2. A compound of claim 1 wherein the glutamic acid hasD-configuration.
 3. A compound of claim 2 wherein R₃ and R₄ are hydroxy.4. A compound of claim 2 wherein R₅ is hydrogen.
 5. A compound of claim3 wherein R₅ is hydrogen.
 6. A compound of claim 1 selected from thegroup consisting of2-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid,2-(L-alanylamino)-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid,2-amino-4-methylene-6-[{N-(N-1-oxo-octadecyl)-L-alanyl)-γ-D-glutamyl}-amino]-heptanedioicacid,2-amino-4-methylene-6-[[N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioicacid and their non-toxic, pharmaceutically acceptable addition saltswith acids and bases.
 7. An immunomodulatory composition comprising animmunomodulatorily effective amount of at least one compound of claim 1and an inert pharmaceutical carrier.
 8. A composition of claim 7 whereinthe glutamic acid has D-configuration.
 9. A composition of claim 8wherein the R₃ and R₄ are hydroxy.
 10. A composition of claim 8 whereinR₅ is hydrogen.
 11. A composition of claim 9 wherein R₅ is hydrogen. 12.A composition of claim 7 wherein the active compound is selected fromthe group consisting of2-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid,2-(L-alanylamino)-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid,2-amino-4-methylene-6-[{N-(N-1-oxo-octadecyl)-L-alanyl)-γ-D-glutamyl}-amino]-heptanedioicacid,2-amino-4-methylene-6-[[N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioicacid and their non-toxic, pharmaceutically acceptable addition saltswith acids and bases.
 13. A method of inducing immunomodulatory activityin warm-blodded animals comprising administering to warm-blooded animalsan immunomodulatorily effective amount of at least one compound ofclaim
 1. 14. A method of claim 13 wherein the glutamic acid hasD-configuration.
 15. A method of claim 14 wherein R₃ and R₄ are hydroxy.16. A method of claim 14 wherein R₅ is hydrogen.
 17. A method of claim15 wherein R₅ is hydrogen.
 18. A method of claim 13 wherein the activecompound is 2-amino-6-(γ-D-glutamylamino)-4-methyleneheptanedioic acid,2-(L-alanylamino)-6-(γ-D-glutamyamino)-4-methyleneheptanedioic acid,2-amino-4-methylene-6-[{N-(N-1-oxo-octadecyl)-L-alanyl)-γ-D-glutamyl}-amino]-heptanedioicacid,2-amino-4-methylene-6-[[N-(1-oxo-octadecyl)-γ-D-glutamyl]-amino]-heptanedioicacid and their non-toxic, pharmaceutically acceptable addition saltswith acids and bases.